1. Field of Invention
The present invention relates to a method of identification and a method of manipulation and, in particular, to a method of bio-identification and a method of manipulation of nanoscale biomolecules by dielectrophoresis.
2. Related Art
Clinically, Candida albicans and Candida tropicalis, which are two common species within Candida, can easily cause infection and disease in humans suffering from hypoimmunity, especially in AIDS patients, patients in ICU (instruction control unit), cancer patients receiving chemotherapy and organ transplant recipients suffering from hypoimmunity. The diseases caused by Candida albicans and Candida tropicalis should be treated as soon as possible after infection. Otherwise, it would lead to Fungemia, which is a complicated disease for treatment and with an extremely high fatality rate. The effectiveness of medication varies with the species of pathologic fungi. Administration with improper medication would lead to fairly poor treatment effect. Severely, improper administration or using common antibiotics frequently would also cause drug resistance of human body. That is, it results in antibiotic resistance in some strains, and, as a result, healing of the aforementioned infection would become very difficult. Hence, it is very crucial to precisely and rapidly detect and identify the species of the pathologic fungi for medication on fungi infection.
However, traditional approach for strain identification by visual inspection requires a long incubation period. For example, yeast has to be incubated for 24˜72 hours, and fungi even have to be incubated for 1˜2 weeks. It is obviously impractical and cannot meet clinical requirements. In recent years, strain identification with DNA-based technique has becoming a common trend in the field of rapid strain identification. However, while current novel DNA chips allow operators to identify multiple target sources simultaneously, it requires a high DNA concentration of the samples. For those samples containing low DNA concentration, it takes about 3˜4 hours to perform 30˜40 cycles of PCR to increase the DNA concentration up to 100 nM˜1 M before hybridization assay. Furthermore, the conventional hybridization mechanism depends mainly on diffusion so that it takes about 3˜4 hours to react. Briefly, it is inevitable to take about 6˜8 hours for strain identification on the DNA chips.
In addition, identification on the DNA chips could be very inaccurate for some strains with sequences containing few base mismatches. It is mainly because the DNA chips require a hybridization solution (4×SSC buffer with a conductivity of 10 mS/cm) with a high ion concentration in operation. However, the high ion concentration in the solution reduces the electrostatic repulsive force between target DNAs and DNA probes so as to prevent DNA separation after hybridization. In other words, even though the target DNAs contain one- to three-base mismatch, they still can adhere tightly on the DNA probes. Therefore, during hybridization, it is usually considered to heat up the DNA chips to a predetermined temperature (about 45˜55□) to increase the selectivity by thermo agitation mechanism in combination with DNA flushing to remove the DNAs containing few base mismatch. Unfortunately, the flushing step usually accidentally removes some hybridized target DNAs and thereby weakens the intensity of fluorescent signal and the sensitivity of the identification.
Another approach of microbial identification is immunoassay, for example ELISA. In general, the procedure of the immunoassay has to modify a substrate with antibodies first to trap specific proteins on the surface of a target first and fix the target on the surface of the substrate. Then, after adding secondary antibodies modified with fluorescent material or color display material to bind the specific proteins on the surface of the target, the identification result can be observed by the fluorescence intensity or color development. However, since two modifications and a period for fixing the target with the antibodies are required, the procedure generally takes approximately 10˜12 hours to complete all the steps. It is relatively time-consuming and has low sensitivity.
Because a dielectrophoretic force has a major inverse relationship to the square of the diameter of a particle in fluid, it is very difficult to manipulate nanoscale small molecules such as DNAs, virus, antibodies and proteins by the dielectrophoretic force. Manipulation of nanoscale biomolecules, for example the coiled DNA with the size of about 1˜10 nm, requires an electric field intensity higher than 108 V/m. That is, it is essential to use nanoelectrodes with the size of 10˜20 nm and a AC voltage of 10˜20 Vpp to generate the dielectrophoretic force considering not to inducing electrolysis at the electrodes. Such small-sized electrodes only can be manufactured in nanoprocess and, in particular, is very difficult to manufacture and low-yield. To this end, no dielectrophoretic microfluidic chip is qualified for manipulation of nanoscale small molecules such as DNAs, virus, antibodies and proteins currently.